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Systemic lupus erythematosus is a complex autoimmune disease with significant morbidity that demands further examination of tolerance-inducing treatments. Short-term treatment of lupus-prone NZB/WF1 mice with combination CTLA4Ig and anti–CD40 ligand, but not single treatment alone, suppresses disease for >6 mo via modulation of B and T cell function while maintaining immune responses to exogenous Ags. Three months after a 2-wk course of combination costimulatory blockade, we found a modest decrease in the number of activated T and B cells in both combination and single-treatment cohorts compared with untreated controls. However, only combination treatment mice showed a 50% decrease in spare respiratory capacity of splenic B and T cells. RNA sequencing and gene set enrichment analysis of germinal center (GC) B cells confirmed a reduction in the oxidative phosphorylation signature in the combination treatment cohort. This cohort also manifested increased expression of BCR-associated signaling molecules and increased phosphorylation of PLCγ in GC B cells after stimulation with anti-IgG and anti-CD40. GC B cells from combination treatment mice also displayed a signature involving remodeling of GPI-linked surface proteins. Accordingly, we found a decrease in cell surface expression of the inhibitory molecule CD24 on class-switched memory B cells from aged NZB/W mice that corrected in the combination treatment cohort. Because both a profound decrease in BCR signaling and remodeled immune cell metabolism enhance loss of tolerance in lupus-prone mice, our findings help to explain the restoration of tolerance observed after short-term combination costimulatory blockade.

BackgroundLong-term kidney allograft survival remains suboptimal. Emerging evidence indicates donor-recipient (D-R) mismatches outside of human leukocyte antigens (HLA) contribute to graft survival but mechanisms remain unclear, specifically for those mismatches within intronic regions.MethodsWe analyzed genome-wide SNP data of D-R pairs from two well-phenotyped kidney transplant cohorts (median follow-up ~1800 days), Genomics of Chronic Allograft Rejection (GoCAR; n=385) and Clinical Trials in Organ Transplantation 1/17 (CTOT1/17; n=146), quantifying genetic mismatches outside of HLA for every D-R pair at variant, gene, and genome-wide scales.ResultsUnbiased genome-wide screen of GoCAR D-R pairs uncovered the LIMS1 locus as the topranked candidate where D-R mismatches associated with death censored graft loss (DCGL). Independent of HLA, a previously unreported relationship between mismatches at a highly linked, intronic haplotype of 30 SNPs was seen as associated with DCGL, with confirmatory association in intra-ancestry D-Rs. Validation testing within the CTOT-01/17 showed similar associations with DCGL. Haplotype D-R mismatches showed a dosage effect, and the introduction of minor alleles in the donor to major allele-carrying recipients showed a greater risk of DCGL. Both the new LIMS1 haplotype and the previously reported LIMS1 SNP rs893403 are expression quantitative trait loci (eQTL) for the gene GCC2 in recipient immune cells, without alterations in GCC2 or LIMS1 coding sequences. Transcriptome enrichment analyses performed on whole blood and within multiple T cell subsets demonstrated significant associations of GCC2 gene, and of either allelic locus, with regulation of TGF-beta-SMAD signaling, implying a role in Treg function and association with rejection.ConclusionsOur analysis unravels intronic non-HLA SNP mismatches within LIMS1 that do not induce protein sequence variation but associate with DCGL. By acting as cis-eQTLs for GCC2 expression, these SNPs modulates TGF-beta signaling and T cell function, associating with immune events and graft outcomes. The findings have clinical implications for risk assessment and individualized therapy in kidney transplant recipients.

Background We have found disruption of expression of major transcriptional regulators of circadian rhythm in the kidneys of several mouse models of lupus nephritis. Here we define the consequence of this disturbance with respect to circadian gene expression and renal homeostatic function in a mouse model of lupus nephritis. Methods Molecular profiling of kidneys from 47 young and 41 nephritic female NZB/W F1 mice was performed at 4 hourly intervals over a 24 h period. Disruption of major circadian transcriptional regulators was confirmed by qPCR. Molecular data was normalized and analyzed for rhythmicity using RAIN analysis. Serum aldosterone and glucose and urine sodium and potassium were measured at 4 hourly intervals in pre-nephritic and nephritic mice and blood pressure was measured every 4 h. Analyses were repeated after induction of complete remission of nephritis using combination cyclophosphamide and costimulatory blockade. Results We show a profound alteration of renal circadian rhythms in mice with lupus nephritis affecting multiple renal pathways. Using Cosinor analysis we identified consequent alterations of renal homeostasis and metabolism as well as blood pressure dipper status. This circadian dysregulation was partially reversed by remission induction therapy. Conclusions Our studies indicate the role of inflammation in causing the circadian disruption and suggest that screening for loss of normal blood pressure dipping should be incorporated into LN management. The data also suggest a potential role for circadian agonists in the treatment of lupus nephritis.

Recent case reports suggest that coronavirus disease 2019 (COVID-19) is associated with collapsing glomerulopathy in African Americans with apolipoprotein L1 gene (APOL1) risk alleles; however, it is unclear whether disease pathogenesis is similar to HIV-associated nephropathy. RNA sequencing analysis of a kidney biopsy specimen from a patient with COVID-19–associated collapsing glomerulopathy and APOL1 risk alleles (G1/G1) revealed similar levels of APOL1 and angiotensin-converting enzyme 2 (ACE2) messenger RNA transcripts as compared with 12 control kidney samples downloaded from the GTEx (Genotype-Tissue Expression) Portal. Whole-genome sequencing of the COVID-19–associated collapsing glomerulopathy kidney sample identified 4 indel gene variants, 3 of which are of unknown significance with respect to chronic kidney disease and/or focal segmental glomerulosclerosis. Molecular profiling of the kidney demonstrated activation of COVID-19–associated cell injury pathways such as inflammation and coagulation. Evidence for direct severe acute respiratory syndrome coronavirus 2 infection of kidney cells was lacking, which is consistent with the findings of several recent studies. Interestingly, immunostaining of kidney biopsy sections revealed increased expression of phospho-STAT3 (signal transducer and activator of transcription 3) in both COVID-19–associated collapsing glomerulopathy and HIV-associated nephropathy as compared with control kidney tissue. Importantly, interleukin 6–induced activation of STAT3 may be a targetable mechanism driving COVID-19–associated acute kidney injury.

Tacrolimus (Tac) is an effective anti-rejection agent in kidney transplantation, but its off-target effects make withdrawal desirable. Although studies indicate that Tac can be safely withdrawn in a subset of kidney transplant recipients, immune mechanisms that underlie successful vs unsuccessful Tac removal are unknown. We performed microarray analyses of peripheral blood mononuclear cells (PBMC) RNA from subjects enrolled in the Clinical Trials in Organ Transplantation-09 study in which we randomized stable kidney transplant recipients to Tac withdrawal or maintenance of standard immunosuppression beginning 6 months after transplant. Eight of 14 subjects attempted but failed withdrawal, while six developed stable graft function for ≥2 years on mycophenolate mofetil plus prednisone. Whereas failed withdrawal upregulated immune activation genes, successful Tac withdrawal was associated with a downregulatory and proapoptotic gene program enriched within T cells. Functional analyses suggested stronger donor-reactive immunity in subjects who failed withdrawal without evidence of regulatory T cell dysfunction. Together, our data from a small, but unique, patient cohort support the conclusion that successful Tac withdrawal is not simply due to absence of donor-reactive immunity but rather is associated with an active immunological process.

Apolipoprotein L1 (APOL1) risk alleles in donor kidneys associate with graft loss, but whether recipient risk allele expression affects transplant outcomes is unclear. To test whether recipient APOL1 risk alleles independently correlate with transplant outcomes, we analyzed genome-wide SNP genotyping data on donors and recipients from 2 kidney transplant cohorts: Genomics of Chronic Allograft Rejection (GOCAR) and Clinical Trials in Organ Transplantation 01/17 (CTOT-01/17). We estimated genetic ancestry (quantified as the proportion of African ancestry, or pAFR) by ADMIXTURE and correlated APOL1 genotypes and pAFR with outcomes. In the GOCAR discovery set, we noted that the number of recipient APOL1 G1/G2 alleles (R-nAPOL1) associated with an increased risk of death-censored allograft loss (DCAL), independent of ancestry (HR = 2.14; P = 0.006), as well as within the subgroup of African American and Hispanic (AA/H) recipients (HR = 2.36; P = 0.003). R-nAPOL1 also associated with an increased risk of any T cell–mediated rejection (TCMR) event. These associations were validated in CTOT-01/17. Ex vivo studies of PMBCs revealed, unexpectedly, high expression levels of APOL1 in activated CD4(+)/CD8(+) T cells and NK cells. We detected enriched immune response gene pathways in risk allele carriers compared with noncarriers on the kidney transplant waitlist and among healthy controls. Our findings demonstrate an immunomodulatory role for recipient APOL1 risk alleles associated with TCMR and DCAL. We believe this finding has broader implications for immune-mediated injury to native kidneys.

Programmed death-ligand 1 (PD-L1) is a strong immunosuppressor that inhibits T-cell activation by binding to its receptor (PD-1) on T-cells. Blocking the PD-L1/PD-1 interaction by monoclonal antibodies (mAbs) is an effective therapy for several cancers via activation of T-cells. Destructive thyroiditis and hypothyroidism often develop during anti-PD-L1 (αPD-L1) therapy but the pathogenic mechanisms are not known. We evaluated in vivo, the role of PD-L1 and its interactions with inflammatory triggers in the development of autoimmune thyroiditis (AIT). NOD.H-2h4 mice were treated with either αPD-L1 or isotype control (CTR) for two weeks and AIT evaluated after six weeks. αPD-L1 treated mice showed significantly higher Abs (p = 6×10e-4) and T-cell responses (p = 0.02) to thyroglobulin (Tg) compared with CTR mice. Serum fT4 levels were lower (p = 0.01) and TSH higher (p = 0.05) in αPD-L1 treated vs. CTR mice. Seven of the eight αPD-L1 treated mice (and none of the CTR mice) developed thyroid lymphocytic infiltration characterized by the presence of the CD4+, CD8+, FoxP3+ T- cells and B220+ B- cells. To gain insight into the pathways regulated by PD-L1 in the thyroid we assessed gene expression in the thyroid of αPD-L1 treated mice using RNA-seq. PD-L1 blockade was associated with upregulation of pathways of lymphocyte activation (p = 1.3×10e-5), lymphocyte proliferation (p = 2×10e-5), chemotaxis (p = 3.8×10e-12), and inflammatory response (p = 7.9×10e-7). Specifically, blocking PD-L1 was associated with upregulation of cytokine/chemokine genes that have a critical role in Th1 differentiation: IFNγ, IL-12, IL-15, CCL1 to CCL5, CD300, CXCL10, CXCL11. Ingenuity Pathway Analysis identified IFNγ and TNF as the top regulators of these activated gene networks. These results indicate that blockade of PD-L1 in thyroid promoted the Th1 mediator, IFNγ, inducing activation and proliferation of thyroidal CD4+ and CD8+ T cells, triggering thyroid tissue damage and AIT. Since PD-L1 mediates immune responses to inflammatory stressors (e.g. viral infections), we evaluated PD-L1 interaction with a known inflammatory trigger of AIT, interferon alpha (IFNα). Treatment with αPD-L1 + IFNα accelerated AIT in NOD.H-2h4 mice that developed high Tg Ab and T-cell responses and thyroidal lymphocytic infiltration only two weeks after treatment. Moreover, thyroiditis was more severe in mice receiving αPD-L1 + IFNα than αPD-L1 alone, manifesting by higher T-cell responses (p = 5×10e-3), and significantly more profound hypothyroidism. These results suggest that PDL-1 has a protective role in the thyroid against inflammatory stressors, and that its blockade exposes the thyroid to inflammatory damage. Therefore, in patients undergoing αPD-L1 therapy, inflammatory conditions (e.g. infections) can be a "second hit" that accelerates the onset of thyroiditis and augments thyroidal inflammation. Presentation: Monday, June 13, 2022 12:00 a.m. - 12:15 a.m.

Context Genetic risk factors play a major role in the pathoetiology of autoimmune thyroid diseases (AITD). So far, only common risk variants have been identified in AITD susceptibility genes. Recently, rare genetic variants have emerged as important contributors to complex diseases, and we hypothesized that rare variants play a key role in the genetic susceptibility to AITD. Objective We aimed to identify new rare variants that are associated with familial AITD. Methods We performed deep sequencing of 3 previously mapped AITD-linked loci (10q, 12q, and 14q) in a dataset of 34 families in which AITD clustered (familial AITD). Results We identified 13 rare variants, located in the inositol polyphosphate multikinase (IPMK) gene, that were associated with AITD (ie, both Graves’ disease [GD] and Hashimoto’s thyroiditis [HT]); 2 rare variants, within the dihydrolipoamide S-succinyltransferase (DLST) and zinc-finger FYVE domain-containing protein (ZFYVE1) genes, that were associated with GD only; and 3 rare variants, within the phosphoglycerate mutase 1 pseudogene 5 (PGAM1P5), LOC105369879, and methionine aminopeptidase 2 (METAP2) genes, that were associated with HT only. Conclusion Our study demonstrates that, in addition to common variants, rare variants also contribute to the genetic susceptibility to AITD. We identified new rare variants in 6 AITD susceptibility genes that predispose to familial AITD. Of these, 3 genes, IPMK, ZFYVE1, and METAP2, are mechanistically involved in immune pathways and have been previously shown to be associated with autoimmunity. These genes predispose to thyroid autoimmunity and may serve as potential therapeutic targets in the future.

ContextHepatitis C virus (HCV) infection is a prevalent disease worldwide. Thyroid dysfunction is one of the most common extrahepatic manifestations of HCV infection. We hypothesized that HCV can directly infect human thyrocytes thereby causing thyroid dysfunction.SettingHuman thyrocytes in primary cell culture, ML-1 human thyroid cell line, and Huh7.5 human hepatocyte cell line were infected with HCV using the Huh7.5JFH1 cell line that releases infectious HCV virions. After infection, the release of new virions, production of proinflammatory cytokines, and expression of miR-122 were evaluated. Ribonucleic acid (RNA) extracted from HCV-infected cells and mock-infected cells was subjected to RNA sequencing and transcriptomic analysis. Ingenuity pathway analysis was used to detect up- and down-regulated pathways.ResultsHuman thyrocytes express major HCV entry factors including CD81, occludin, claudin-1, and scavenger receptor class B1. Viral infection of thyroid cells was confirmed by detection of HCV core protein in supernatants and negative-sense HCV RNA in cell lysates. HCV infection of thyrocytes induced the production of the chemokine CXCL-8 and the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and significantly increased the expression of miR-122. Moreover, HCV infection of thyrocytes decreased expression of the thyroid peroxidase and thyroglobulin genes and increased expression of the deiodinase 2 gene. The top upregulated pathways in HCV-infected thyrocytes were immune pathways and metabolic pathways, while infected hepatocytes upregulated lipid and glucose metabolism pathways as previously reported.ConclusionsHCV infection may induce thyroid dysfunction by different mechanisms including direct infection of thyrocytes leading to activation of inflammatory pathways and upregulation of miR-122. These findings support a general mechanism for viral induction of autoimmunity through direct infection of target tissues.

HIV-associated nephropathy (HIVAN) is a rapidly progressive kidney disease that is caused by HIV infection of renal epithelial cells with subsequent expression of viral genes, including vpr. Antiretroviral therapy ameliorates HIVAN without eradicating HIV from the kidneys and the mechanism by which it protects kidneys is poorly understood. Since HIV protease inhibitors have “off target” cellular effects, we studied whether darunavir, the most commonly prescribed protease inhibitor, protects kidneys from HIV-induced injury via mechanisms independent of HIV protease and viral replication. Renal epithelial cells were transduced with lentiviruses encoding HIV (lacking protease and reverse transcriptase), Vpr, or vector control. Darunavir attenuated HIV and Vpr-induced activation of Stat3, Src, Erk, and cytokines, which are critical for HIVAN pathogenesis. We then studied HIV-transgenic mice, which develop HIVAN in the absence of HIV protease or reverse transcriptase. Mice were treated with darunavir, zidovudine, darunavir + zidovudine, or control. Darunavir and darunavir + zidovudine reduced albuminuria and histologic kidney injury and normalized expression of dysregulated proteins. RNA-seq analyses demonstrated that darunavir suppressed HIV-induced upregulation of immune response genes in human kidney cells. These data demonstrate that darunavir protects against HIV-induced renal injury via mechanisms that are independent of inhibition of HIV protease.

Emerging evidence of crosstalk between glomerular cells in pathological settings provides opportunities for novel therapeutic discovery. Here we investigated underlying mechanisms of early events leading to filtration barrier defects of podocyte and glomerular endothelial cell crosstalk in the mouse models of primary podocytopathy (podocyte specific transforming growth factor-β receptor 1 signaling activation) or Adriamycin nephropathy. We found that glomerular endothelial surface layer degradation and albuminuria preceded podocyte foot process effacement. These abnormalities were prevented by endothelin receptor-A antagonism and mitochondrial reactive oxygen species scavenging. Additional studies confirmed increased heparanase and hyaluronoglucosaminidase gene expression in glomerular endothelial cells in response to podocyte-released factors and to endothelin-1. Atomic force microscopy measurements showed a significant reduction in the endothelial surface layer by endothelin-1 and podocyte-released factors, which could be prevented by endothelin receptor-A but not endothelin receptor-B antagonism. Thus, our studies provide evidence of early crosstalk between activated podocytes and glomerular endothelial cells resulting in loss of endothelial surface layer, glomerular endothelial cell injury and albuminuria. Hence, activation of endothelin-1-endothelin receptor-A and mitochondrial reactive oxygen species contribute to the pathogenesis of primary podocytopathies in experimental focal segmental glomerulosclerosis.

BackgroundInterstitial fibrosis, tubular atrophy, and inflammation are major contributors to renal allograft failure. Here we seek an objective, quantitative pathological assessment of these lesions to improve predictive utility.MethodsWe constructed a deep-learning-based pipeline recognizing normal vs. abnormal kidney tissue compartments and mononuclear leukocyte (MNL) infiltrates from Periodic acid-Schiff (PAS) stained slides of transplant biopsies (training: n=60, testing: n=33) that quantified pathological lesions specific for interstitium, tubules and MNL infiltration. The pipeline was applied to 789 whole slide images (WSI) from baseline (n=478, pre-implantation) and 12-month post-transplant (n=311) protocol biopsies in two independent cohorts (GoCAR: 404 patients, AUSCAD: 212 patients) of transplant recipients to correlate composite lesion features with graft loss.ResultsOur model accurately recognized kidney tissue compartments and MNLs. The digital features significantly correlated with Banff scores, but were more sensitive to subtle pathological changes below the thresholds in Banff scores. The Interstitial and Tubular Abnormality Score (ITAS) in baseline samples was highly predictive of 1-year graft loss (p=2.8e-05), while a Composite Damage Score (CDS) in 12-month post-transplant protocol biopsies predicted later graft loss (p=7.3e-05). ITAS and CDS outperformed Banff scores or clinical predictors with superior graft loss prediction accuracy. High/intermediate risk groups stratified by ITAS or CDS also demonstrated significantly higher incidence of eGFR decline and subsequent graft damage.ConclusionsThis deep-learning approach accurately detected and quantified pathological lesions from baseline or post-transplant biopsies, and demonstrated superior ability for prediction of posttransplant graft loss with potential application as a prevention, risk stratification or monitoring tool.

Dachshund homolog 1 (DACH1), a key cell-fate determinant, regulates transcription by DNA sequence-specific binding. We identified diminished Dach1 expression in a large-scale screen for mutations that convert injury-resistant podocytes into injury-susceptible podocytes. In diabetic kidney disease (DKD) patients, podocyte DACH1 expression levels are diminished, a condition that strongly correlates with poor clinical outcomes. Global Dach1 KO mice manifest renal hypoplasia and die perinatally. Podocyte-specific Dach1 KO mice, however, maintain normal glomerular architecture at baseline, but rapidly exhibit podocyte injury after diabetes onset. Furthermore, podocyte-specific augmentation of DACH1 expression in mice protects from DKD. Combined RNA sequencing and in silico promoter analysis reveal conversely overlapping glomerular transcriptomic signatures between podocyte-specific Dach1 and Pax transactivation-domain interacting protein (Ptip) KO mice, with upregulated genes possessing higher-than-expected numbers of promoter Dach1-binding sites. PTIP, an essential component of the activating histone H3 lysine 4 trimethylation (H3K4Me3) complex, interacts with DACH1 and is recruited by DACH1 to its promoter-binding sites. DACH1-PTIP recruitment represses transcription and reduces promoter H3K4Me3 levels. DACH1 knockdown in podocytes combined with hyperglycemia triggers target gene upregulation and increases promoter H3K4Me3. These findings reveal that in DKD, diminished DACH1 expression enhances podocyte injury vulnerability via epigenetic derepression of its target genes.

Apolipoprotein L1 (APOL1) risk alleles in donor kidneys associate with graft loss but whether recipient risk allele expression impacts kidney transplant outcomes is unclear. To test whether recipient APOL1 allelic variants independently correlate with transplant outcomes, we analyzed genome-wide SNP genotyping data of donors and recipients from two kidney transplant cohorts, Genomics of Chronic Allograft Rejection (GOCAR) and Clinical Trials in Organ Transplantation 1/17 (CTOT1/17). We estimated genetic ancestry (quantified as proportion of African ancestry or pAFR) by ADMIXTURE and correlated APOL1 genotypes and pAFR with outcomes. In the GOCAR discovery set, we observed that the number of recipient APOL1 G1/G2 alleles (R-nAPOL1) associated with increased risk of death-censored allograft loss (DCAL), independent of genetic ancestry (HR = 2.14; P = 0.006), and within the subgroup of African American and Hispanic (AA/H) recipients (HR = 2.36; P = 0.003). R-nAPOL1 also associated with increased risk of any T cell-mediated rejection (TCMR) event. Analysis of the CTOT cohort validated these associations. Ex vivo studies of peripheral blood mononuclear cells revealed unanticipated high APOL1 expression in activated CD4+/CD8+ T cells and natural killer cells. We detected enriched immune response gene pathways in G1/G2 allele carriers vs. non-carriers among patients on the kidney waitlist and healthy controls. Together our findings highlight a previously unrecognized contribution of recipient APOL1 risk alleles to renal allograft outcomes. This immunomodulatory role has broader implications for immune mediated injury to native kidneys.

Chronic kidney disease (CKD) is a complex disease in which the renal function is compromised chronically. Many studies have indicated the crosstalk between the tubule and the glomerulus in CKD progression. However, our understanding of the interaction of tubular and glomerular injury remains incomplete. In this study, we applied a meta-analysis approach on the transcriptome of the tubules and glomeruli of CKD patients to identify differentially expressed genes (DEGs) signature. Functional analysis of pathways and Gene Ontology found that tubular DEGs were mainly involved in cell assembly and remodeling, glomerular DEGs in cell proliferation and apoptosis, and overlapping DEGs mainly in immune response. Correlation analysis was performed to identify the associated DEGs in the tubules and glomeruli. Secreted protein comparison and verification experiments indicated that WFDC2 from the tubule could downregulate PEX19 mRNA and protein levels at the glomeruli in diabetic kidney disease (DKD). This study revealed the distinctive pathways of the tubules and glomeruli and identified interacted genes during CKD progression.

Acute rejection (AR) in renal transplantation is an established risk factor for reduced allograft survival. Molecules with regulatory control among immune pathways of AR that are inadequately suppressed, despite standard-of-care immunosuppression, could serve as important targets for therapeutic manipulation to prevent rejection. Here, an integrative, network-based computational strategy incorporating gene expression and genotype data of human renal allograft biopsy tissue was applied, to identify the master regulators — the key driver genes (KDGs) — within dysregulated AR pathways. A 982–meta-gene signature with differential expression in AR versus non-AR was identified from a meta-analysis of microarray data from 735 human kidney allograft biopsy samples across 7 data sets. Fourteen KDGs were derived from this signature. Interrogation of 2 publicly available databases identified compounds with predicted efficacy against individual KDGs or a key driver–based gene set, respectively, which could be repurposed for AR prevention. Minocycline, a tetracycline antibiotic, was chosen for experimental validation in a murine cardiac allograft model of AR. Minocycline attenuated the inflammatory profile of AR compared with controls and when coadministered with immunosuppression prolonged graft survival. This study demonstrates that a network-based strategy, using expression and genotype data to predict KDGs, assists target prioritization for therapeutics in renal allograft rejection.

Donor-recipient (D-R) differences at human leukocyte antigen (HLA) loci are currently incorporated into organ sharing, allocation and immunosuppression decisions. However, while acute rejection episodes have substantially diminished, progressive histologic damage occurs in allografts and improved long-term survival remains an unrealized goal among kidney recipients. Here we tested the hypothesis that non-HLA dependent, genome-wide D-R genetic differences could contribute to unchecked alloimmunity with histologic and functional consequences, culminating in long-term allograft failure. Genome-wide single nucleotide polymorphism (SNP) array data, excluding the HLA region, was utilized from 385 transplants to study the role of D-R differences upon serial histology and allograft survival. ADMIXTURE analysis was performed to quantitatively estimate ancestry in each D-R pair and PLINK was used to estimate the proportion of genome-shared identity-by-descent (pIBD) between D-R pairs. Subsequently, quantitative measures of recipient ancestry based on non-HLA SNPs was associated with death-censored allograft survival in adjusted Cox models. In D-R pairs of similar ancestry, pIBD was significantly associated with allograft survival independent of HLA mismatches in 224 transplants. Surprisingly, pIBD and recipient ancestry were not associated with clinical or subclinical rejection at any time post-transplant. Significantly, in multivariable analysis, pIBD inversely correlated with vascular intimal fibrosis in 160 biopsies obtained less than one year which in turn was significantly associated with allograft survival. Thus, our novel data show that non-HLA D-R differences associate with early vascular intimal fibrosis and allograft survival.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Background We previously showed that the presence of a CKD-associated locus in SHROOM3 in a donor kidney results in increased expression of SHROOM3 (an F-actin–binding protein important for epithelial morphogenesis, via rho-kinase [ROCK] binding); this facilitates TGF-b signaling and allograft fibrosis. However, other evidence suggests Shroom3 may have a protective role in glomerular development. Methods We used human data, Shroom3 knockdown podocytes, and inducible shRNA-mediated knockdown mice to study the role of Shroom3 in adult glomeruli. Results Expression data from the Nephroseq database showed glomerular and nonglomerular SHROOM3 had opposing associations with renal function in CKD biopsy samples. In human allografts, homozygosity at rs17319721, the SHROOM3 locus linked with lower GFR, was associated with reduced albuminuria by 2 years after transplant. Although our previous data showed reduced renal fibrosis with tubular Shroom3 knockdown, this study found that glomerular but not tubular Shroom3 knockdown induced albuminuria. Electron microscopy revealed diffuse foot process effacement, and glomerular RNA-sequencing showed enrichment of tyrosine kinase signaling and podocyte actin cytoskeleton pathways in knockdown mice. Screening SHROOM3-interacting proteins identified FYN (a src-kinase) as a candidate.We confirmed the interaction of endogenous SHROOM3 with FYN in human podocytes via a critical Src homology 3–binding domain, distinct from its ROCK-binding domain. Shroom3-Fyn interaction was required in vitro and in vivo for activation of Fyn kinase and downstream nephrin phosphorylation in podocytes. SHROOM3 knockdown altered podocyte morphology, cytoskeleton, adhesion, and migration. Conclusions We demonstrate a novel mechanism that may explain SHROOM3’s dichotomous associations in glomerular versus nonglomerular compartments in CKD

Induction of proinflammatory T cell immunity is augmented by innate dendritic cell (DC) maturation commonly initiated by TLR signaling. We demonstrate that ligation of TLR3, TLR4, and TLR9 induces murine DC production of complement components and local production of the anaphylatoxin C5a. In vitro, ex vivo, and in vivo analyses show that TLR-induced DC maturation, as assessed by surface phenotype, expression profiling by gene array, and functional ability to stimulate T cell responses, requires autocrine C3a receptor and C5a receptor (C3ar1/C5ar1) signaling. Studies using bone marrow chimeric animals and Foxp3-GFP/ERT2-Cre/dTomato fate-mapping mice show that TLR-initiated DC autocrine C3ar1/C5ar1 signaling causes expansion of effector T cells and instability of regulatory T cells and contributes to T cell–dependent transplant rejection. Together, our data position immune cell–derived complement production and autocrine/paracrine C3ar1/C5ar1 signaling as crucial intermediary processes that link TLR stimulation to DC maturation and the subsequent development of effector T cell responses.

Renal fibrosis is the common pathway of progression for patients with CKD and chronic renal allograft injury (CAI), but the underlying mechanisms remain obscure. We performed a meta-analysis in human kidney biopsy specimens with CAI, incorporating data available publicly and from our Genomics of Chronic Renal Allograft Rejection study. We identified an Src family tyrosine kinase, hematopoietic cell kinase ( Hck ), as upregulated in allografts in CAI. Querying the Kinase Inhibitor Resource database revealed that dasatinib, a Food and Drug Administration–approved drug, potently binds Hck with high selectivity. In vitro , Hck overexpression activated the TGF-β/Smad3 pathway, whereas HCK knockdown inhibited it. Treatment of tubular cells with dasatinib reduced the expression of Col1a1 . Dasatinib also reduced proliferation and α-SMA expression in fibroblasts. In a murine model with unilateral ureteric obstruction, pretreatment with dasatinib significantly reduced the upregulation of profibrotic markers, phosphorylation of Smad3, and renal fibrosis observed in kidneys pretreated with vehicle alone. Dasatinib treatment also improved renal function, reduced albuminuria, and inhibited expression of profibrotic markers in animal models with lupus nephritis and folic acid nephropathy. These data suggest that Hck is a key mediator of renal fibrosis and dasatinib could be developed as an antifibrotic drug.

Commonly available clinical parameters fail to predict early acute cellular rejection (EAR, occurring within 6 months after transplant), a major risk factor for graft loss after kidney transplantation. We performed whole-blood RNA sequencing at the time of transplant in 235 kidney transplant recipients enrolled in a prospective cohort study (Genomics of Chronic Allograft Rejection [GoCAR]) and evaluated the relationship of pretransplant transcriptomic profiles with EAR. EAR was associated with downregulation of NK and CD8(+) T cell gene signatures in pretransplant blood. We identified a 23-gene set that predicted EAR in the discovery (n = 81, and AUC = 0.80) and validation (n = 74, and AUC = 0.74) sets. Exclusion of recipients with 5 or 6 HLA donor mismatches increased the AUC to 0.89. The risk score derived from the gene set was also significantly associated with acute cellular rejection after 6 months, antibody-mediated rejection and/or de novo donor-specific antibodies, and graft loss in a cohort of 154 patients, combining the validation set and additional GoCAR patients with surveillance biopsies between 6 and 24 months (n = 80) posttransplant. This 23-gene set is a potentially important new tool for determination of the recipient’s immunological risk before kidney transplantation, and facilitation of an individualized approach to immunosuppressive therapy.

Here we assessed the diagnostic value of a quantitative multiparametric magnetic resonance imaging (mpMRI) protocol for evaluation of renal allograft dysfunction with fibrosis. Twenty-seven renal transplant patients, including 15 with stable functional allografts (eGFR mean 71.5 ml/min/1.73m(2)), and 12 with chronic dysfunction/established fibrosis (eGFR mean 30.1 ml/min/1.73m(2)) were enrolled in this prospective single-center study. Sixteen of the patients had renal biopsy (mean 150 days) before the MRI. All patients underwent mpMRI at 1.5T including intravoxel-incoherent motion diffusion-weighted imaging, diffusion tensor imaging, blood oxygen level dependent (BOLD R(2)*) and T(1) quantification. True diffusion D, pseudodiffusion D*, perfusion fraction PF, apparent diffusion coefficient (ADC), fractional anisotropy (FA), R(2)* and T(1) were calculated for cortex and medulla. ΔT(1) was calculated as (100x(T(1) Cortex-T(1) Medulla)/T(1) Cortex). Test-retest repeatability and inter-observer reproducibility were assessed in four and ten patients, respectively. mpMRI parameters had substantial test-retest and interobserver repeatability (coefficient of variation under 15%), except for medullary PF and D* (coefficient of variation over 25%). Cortical ADC, D, medullary ADC and ΔT(1) were all significantly decreased, while cortical T(1) was significantly elevated in fibrotic allografts. Cortical T(1) showed positive correlation to the Banff fibrosis and tubular atrophy scores. The combination of ΔT(1) and cortical ADC had excellent cross-validated diagnostic performance for detection of chronic dysfunction with fibrosis. Cortical ADC and T(1) had good performance for predicting eGFR decline at 18 months (4 or more ml/min/1.73m(2)/year). Thus, the combination of cortical ADC and T(1) measurements show promising results for the non-invasive assessment of renal allograft histology and outcomes.

To identify the factors mediating the progression of diabetic nephropathy (DN), we performed RNA sequencing of kidney biopsy samples from patients with early DN, advanced DN, and normal kidney tissue from nephrectomy samples. A set of genes that were upregulated at early but downregulated in late DN were shown to be largely renoprotective, which included genes in the retinoic acid pathway and glucagon-like peptide 1 receptor. Another group of genes that were downregulated at early but highly upregulated in advanced DN consisted mostly of genes associated with kidney disease pathogenesis, such as those related to immune response and fibrosis. Correlation with estimated glomerular filtration rate (eGFR) identified genes in the pathways of iron transport and cell differentiation to be positively associated with eGFR, while those in the immune response and fibrosis pathways were negatively associated. Correlation with various histopathological features also identified the association with the distinct gene ontological pathways. Deconvolution analysis of the RNA sequencing data set indicated a significant increase in monocytes, fibroblasts, and myofibroblasts in advanced DN kidneys. Our study thus provides potential molecular mechanisms for DN progression and association of differential gene expression with the functional and structural changes observed in patients with early and advanced DN.

Type 1 diabetes (T1D) is caused by autoimmune destruction of pancreatic β cells. Mounting evidence supports a central role for β cell alterations in triggering the activation of self-reactive T cells in T1D. However, the early deleterious events that occur in β cells, underpinning islet autoimmunity, are not known. We hypothesized that epigenetic modifications induced in β cells by inflammatory mediators play a key role in initiating the autoimmune response. We analyzed DNA methylation (DNAm) patterns and gene expression in human islets exposed to IFN-α, a cytokine associated with T1D development. We found that IFN-α triggers DNA demethylation and increases expression of genes controlling inflammatory and immune pathways. We then demonstrated that DNA demethylation was caused by upregulation of the exoribonuclease, PNPase old-35 (PNPT1), which caused degradation of miR-26a. This in turn promoted the upregulation of ten-eleven translocation 2 (TET2) enzyme and increased 5-hydroxymethylcytosine levels in human islets and pancreatic β cells. Moreover, we showed that specific IFN-α expression in the β cells of IFNα–INS1(CreERT2) transgenic mice led to development of T1D that was preceded by increased islet DNA hydroxymethylation through a PNPT1/TET2–dependent mechanism. Our results suggest a new mechanism through which IFN-α regulates DNAm in β cells, leading to changes in expression of genes in inflammatory and immune pathways that can initiate islet autoimmunity in T1D.

CD4+ follicular helper T (Tfh) cells are specialized providers of T cell help to B cells and can function as pathogenic mediators of murine antibody-dependent chronic graft-versus-host disease (GvHD). Using a parent→F1 model of lupus-like chronic GvHD, in which Tfh cell and germinal center (GC) B cell differentiation occurs over 14 days, we demonstrate that absence of CD4+ T cell-expressed C5a receptor 1 (C5ar1) or pharmacological C5aR1 blockade abrogated generation/expansion of Tfh cells, GC B cells, and autoantibodies. In a Tfh cell-dependent model of chronic GvHD manifested by bronchiolitis obliterans syndrome (BOS), C5aR1 antagonism initiated in mice with established disease ameliorated BOS and abolished the associated differentiation of Tfh and GC B cells. Guided by RNA-sequencing data, mechanistic studies performed using murine and human T cells showed that C5aR1 signaling amplifies IL-6-dependent expression of the transcription factor c-MAF and the cytokine IL-21 via phosphorylating phosphokinase B (AKT) and activating the mammalian target of rapamycin (mTOR). In addition to linking C5aR1-initiated signaling to Tfh cell differentiation, our findings suggest that C5aR1 may be a useful therapeutic target for prevention and/or treatment of individuals with Tfh cell-dependent diseases, including those chronic GvHD patients who have anti-host reactive antibodies.

Aging of established antiviral T cell memory fosters a series of progressive adaptations that paradoxically improve rather than compromise protective CD8+T cell immunity. We now provide evidence that this gradual evolution, the pace of which is contingent on the precise context of the primary response, also impinges on the molecular mechanisms that regulate CD8+ memory T cell (CD8+TM) homeostasis. Over time, CD8+TM become more resistant to apoptosis and acquire enhanced cytokine responsiveness without adjusting their homeostatic proliferation rates; concurrent metabolic adaptations promote increased CD8+TM quiescence and fitness but also impart the re-acquisition of a partial effector-like metabolic profile; and a gradual redistribution of aging CD8+TM from blood and nonlymphoid tissues to lymphatic organs results in CD8+TM accumulations in bone marrow, splenic white pulp and particularly lymph nodes. Altogether, these data demonstrate how temporal alterations of fundamental homeostatic determinants converge to render aged CD8+TM poised for greater recall responses.ABBREVIATIONSATadoptive transferATGLadipose triglyceride lipaseBMPblood and marginated poolFA, FAO, FAS, FASNfatty acid, fatty acid oxidation, fatty acid synthesis, fatty acid synthaseFSC, SSCforward scatter, side scatterGMFIgeometric mean of fluorescence intensityGP, NPglycoprotein, nucleoproteinGSEAgene set enrichment analysisGSHglutathioneIo, IIoprimary, secondaryKEGGKyoto Encyclopedia of Genes and GenomesLALlysosomal acid lipase (LIPA)LCMVlymphocytic choriomeningitis virusNESnormalized enrichment scoreNLTsnonlymphoid tissuesO, Yold, youngOxPhosoxidative phosphorylationp14 cellsTCRtg CD8+T cells specific for the LCMV-GP33-41 determinantRP, WPred pulp, white pulp (spleen)T cell subsetsTEeffector T cellsTCMcentral memory T cells (CD62Lhi)TEMeffector memory T cells (CD62Llo)TEMRAterminally differentiated CD45RA+ effector memory T cells (human)TMmemory T cellsTMPmemory-phenotype T cells (CD44hi)TNnaïve T cells (CD44lo)TRMresident memory T cells (CD69/CD103-enriched)TCRtgT cell receptor transgenicTSLPThymic stromal lymphopoietin

In the article cited above, the Gene Expression Omnibus (GEO) accession number for the RNA-seq data set …

Background In kidney transplant recipients, surveillance biopsies can reveal, despite stable graft function, histologic features of acute rejection and borderline changes that are associated with undesirable graft outcomes. Noninvasive biomarkers of subclinical acute rejection are needed to avoid the risks and costs associated with repeated biopsies. Methods We examined subclinical histologic and functional changes in kidney transplant recipients from the prospective Genomics of Chronic Allograft Rejection (GoCAR) study who underwent surveillance biopsies over 2 years, identifying those with subclinical or borderline acute cellular rejection (ACR) at 3 months (ACR-3) post-transplant. We performed RNA sequencing on whole blood collected from 88 individuals at the time of 3-month surveillance biopsy to identify transcripts associated with ACR-3, developed a novel sequencing-based targeted expression assay, and validated this gene signature in an independent cohort. Results Study participants with ACR-3 had significantly higher risk than those without ACR-3 of subsequent clinical acute rejection at 12 and 24 months, faster decline in graft function, and decreased graft survival in adjusted Cox analysis. We identified a 17-gene signature in peripheral blood that accurately diagnosed ACR-3, and validated it using microarray expression profiles of blood samples from 65 transplant recipients in the GoCAR cohort and three public microarray datasets. In an independent cohort of 110 transplant recipients, tests of the targeted expression assay on the basis of the 17-gene set showed that it identified individuals at higher risk of ongoing acute rejection and future graft loss. Conclusions Our targeted expression assay enabled noninvasive diagnosis of subclinical acute rejection and inflammation in the graft and may represent a useful tool to risk-stratify kidney transplant recipients.

The importance of BET protein BRD4 in gene transcription is well recognized through the study of chemical modulation of its characteristic tandem bromodomain (BrD) binding to lysine-acetylated histones and transcription factors. However, while monovalent inhibition of BRD4 by BET BrD inhibitors such as JQ1 blocks growth of hematopoietic cancers, it is much less effective generally in solid tumors. Here, we report a thienodiazepine-based bivalent BrD inhibitor, MS645, that affords spatially constrained tandem BrD inhibition and consequently sustained repression of BRD4 transcriptional activity in blocking proliferation of solid-tumor cells including a panel of triple-negative breast cancer (TNBC) cells. MS645 blocks BRD4 binding to transcription enhancer/mediator proteins MED1 and YY1 with potency superior to monovalent BET inhibitors, resulting in down-regulation of proinflammatory cytokines and genes for cell-cycle control and DNA damage repair that are largely unaffected by monovalent BrD inhibition. Our study suggests a therapeutic strategy to maximally control BRD4 activity for rapid growth of solid-tumor TNBC cells.

Dystonia is characterized by involuntary muscle contractions. Its many forms are genetically, phenotypically and etiologically diverse and it is unknown whether their pathogenesis converges on shared pathways. Mutations in THAP1 [THAP (Thanatos-associated protein) domain containing, apoptosis associated protein 1], a ubiquitously expressed transcription factor with DNA binding and protein-interaction domains, cause dystonia, DYT6. There is a unique, neuronal 50-kDa Thap1-like immunoreactive species, and Thap1 levels are auto-regulated on the mRNA level. However, THAP1 downstream targets in neurons, and the mechanism via which it causes dystonia are largely unknown. We used RNA-Seq to assay the in vivo effect of a heterozygote Thap1 C54Y or ΔExon2 allele on the gene transcription signatures in neonatal mouse striatum and cerebellum. Enriched pathways and gene ontology terms include eIF2α Signaling, Mitochondrial Dysfunction, Neuron Projection Development, Axonal Guidance Signaling, and Synaptic LongTerm Depression, which are dysregulated in a genotype and tissue-dependent manner. Electrophysiological and neurite outgrowth assays were consistent with those enrichments, and the plasticity defects were partially corrected by salubrinal. Notably, several of these pathways were recently implicated in other forms of inherited dystonia, including DYT1. We conclude that dysfunction of these pathways may represent a point of convergence in the pathophysiology of several forms of inherited dystonia., Author summary Dystonia is a brain disorder that causes disabling involuntary muscle contractions and abnormal postures. Mutations in THAP1, a zinc-finger transcription factor, cause DYT6, but its neuronal targets and functions are unknown. In this study, we sought to determine the effects of Thap1C54Y and ΔExon2 alleles on the gene transcription signatures at postnatal day 1 (P1) in the mouse striatum and cerebellum in order to correlate function with specific genes or pathways. Our unbiased transcriptomics approach showed that Thap1 mutants revealed multiple signaling pathways involved in neuronal plasticity, axonal guidance, and oxidative stress response, which are also present in other forms of dystonia, particularly DYT1. We conclude that dysfunction of these pathways may represent a point of convergence on the pathogenesis of unrelated forms of inherited dystonia.

Fibrosis underlies the loss of renal function in patients with chronic kidney disease (CKD) and in kidney transplant recipients with chronic allograft nephropathy (CAN). Here, we studied the effect of an intronic SNP in SHROOM3, which has previously been linked to CKD, on the development of CAN in a prospective cohort of renal allograft recipients. The presence of the rs17319721 allele at the SHROOM3 locus in the donor correlated with increased SHROOM3 expression in the allograft. In vitro, we determined that the sequence containing the risk allele at rs17319721 is a transcription factor 7-like 2-dependent (TCF7L2-dependent) enhancer element that functions to increase SHROOM3 transcription. In renal tubular cells, TGF-β1 administration upregulated SHROOM3 expression in a β-catenin/TCF7L2-mediated manner, while SHROOM3 in turn facilitated canonical TGF-β1 signaling and increased α1 collagen (COL1A1) expression. Inducible and tubular cell-specific knockdown of Shroom3 markedly abrogated interstitial fibrosis in mice with unilateral ureteric obstruction. Moreover, SHROOM3 expression in allografts at 3 months after transplant and the presence of the SHROOM3 risk allele in the donor correlated with increased allograft fibrosis and with reduced estimated glomerular filtration rate at 12 months after transplant. Our findings suggest that rs17319721 functions as a cis-acting expression quantitative trait locus of SHROOM3 that facilitates TGF-β1 signaling and contributes to allograft injury.

The aim of this study was to investigate the role of infiltrating macrophages in renal allograft fibrosis. Forty-six protocol renal allograft biopsies obtained 1 year after transplantation were stained with Sirius red to quantify fibrosis and double stained with CD68 and CD206 to identify the proportion of alternatively activated (M2) macrophages. Biopsies were analyzed for gene expression by microarray, which was correlated with macrophage infiltration and the severity of fibrosis. The number of infiltrating CD68+ cells strongly correlated with the percentage of interstitial fibrosis (r = 0.73, p

Type 1 diabetes (T1D) shows ~ 40% concordance rate in monozygotic twins (MZ) suggesting a role for environmental factors and/or epigenetic modifications in the etiology of the disease. The aim of our study was to dissect the contribution of epigenetic factors, particularly, DNA methylation (DNAm), to the incomplete penetrance of T1D. We performed DNAm profiling in lymphocyte cell lines from 3 monozygotic (MZ) twin pairs discordant for T1D and 6 MZ twin pairs concordant for the disease using HumanMethylation27 BeadChip. This assay assesses the methylation state of 27,578 CpG sites, mostly located within proximal promoter regions. We identified 88 CpG sites displaying significant methylation changes in all T1D-discordant MZ twin pairs. Functional annotation of the genes with distinct CpG methylation profiles in T1D samples showed differential DNAm of immune response and defense response pathways between affected and unaffected twins. Integration of DNAm data with GWAS data mapped several known T1D associated genes, HLA, INS, IL-2RB, CD226, which showed significant differences in DNAm between affected and unaffected of twins. Our findings suggest that abnormalities of DNA methylation patterns, known to regulate gene transcription, may be involved in the pathogenesis of T1D.

Increased polyamine synthesis is known to play an important role in prostate cancer. We aimed to explore its functional significance in prostate tumor initiation and its link to androgen receptor (AR) signaling. For this purpose, we generated a new cell line derived from normal epithelial prostate cells (RWPE-1) with overexpression of ornithine decarboxylase (ODC) and used it for in vitro and in vivo experiments. We then comprehensively analyzed the expression of the main metabolic enzymes of the polyamine pathway and spermine abundance in 120 well-characterized cases of human prostate cancer and high-grade prostate intraepithelial neoplasia (HGPIN). Herein, we show that the ODC-overexpressing prostate cells underwent malignant transformation, revealing that ODC is sufficient for de novo tumor initiation in 94% of injected mice. This oncogenic capacity was acquired through alteration of critical signaling networks, including AR , EIF2 , and mTOR/MAPK . RNA silencing experiments revealed the link between AR signaling and polyamine metabolism. Human prostate cancers consistently demonstrated up-regulation of the main polyamine enzymes analyzed (ODC, polyamine oxidase, and spermine synthase) and reduction of spermine. This phenotype was also dominant in HGPIN, rendering it a new biomarker of malignant transformation. In summary, we report that ODC plays a key role in prostate tumorigenesis and that the polyamine pathway is altered as early as HGPIN.